1. Field of the Invention
The present invention relates generally to the Jak family of kinases and their role in the cellular response to the binding of cytokines to their respective receptors. The invention relates more specifically to the cytokine-induced activation of at least one member of a Jak kinase family, to the identification of interactions between specific cytokines and members of the Jak kinase family, and to compounds, compositions and methods relating to the regulation of this interaction.
2. Description of the Background Art
The growth, differentiation and function of eukaryotic cells is regulated in large part by extracellular factors, referred to generally as cytokines herein. These cytokines induce cellular responses by binding to their respective receptors. The receptors for cytokines fall into two major families, the cytokine receptor superfamily and the tyrosine kinase receptor superfamily.
Receptors belonging to the tyrosine kinase receptor superfamily are characterized by the presence of an identifiable cytoplasmic tyrosine kinase domain involved in the transduction of the cytokine-receptor binding signal. Members of this receptor family have been further classified into three structural subgroups (Yarden et al., Ann. Rev. Biochem. 57: 443-478 (1988). Members of the first subgroup are characterized as monomeric with two cysteine rich sequence repeat regions within their extracellular domains and include, e.g., the receptor for epidermal growth factor (EGF) and TGF-α (see, e.g., Ullrich et al., Nature 309: 418-425 (1984)). Members of the second subgroup are characterized as functioning as heterotetramers and include the receptors for insulin (Ullrich, supra, (1985); Ebina et al., Cell 40: 747-758 (1985)) and insulin-like growth factor-1 (IGF-1) (Ullrich et al., EMBO J. 5:2503-2512 (1986)). Members of the third subgroup are characterized by the presence of conserved repeat structures and the interruption of their catalytic domains by long (77-107 amino acids) insertion sequences. This third subgroup includes, e.g., receptors for platelet-derived growth factor (PDGF-R) (Yarden et al., Nature 323: 226-232 (1986)) and the colony stimulating growth factor (CSF-1) (Sherr et al., Cell 41: 665-676 (1985)).
Receptors belonging to the cytokine receptor superfamily are characterized by the presence of four positionally conserved cysteines and a WSXWS (SEQ ID No. 1) motif in the extracellular domain. The family is also characterized by variably sized cytoplasmic domains that show very limited sequence similarity and which do not contain identifiable motifs that might indicate the signal transducing mechanisms. Members of the cytokine receptor superfamily include the hematopoietic growth factor receptors, receptors for growth hormone, the prolactin receptor, ciliary neurotrophic factor and others (Bazan, Science 257:410-413 (1992)). The receptors for interferon, although more distantly related, have been speculated to have evolved from a progenitor common to this receptor superfamily.
In spite of the lack of catalytic domains, considerable evidence suggests that signal transduction of members of the cytokine receptor superfamily involves tyrosine phosphorylation (Miyajima et al., Annu. Rev. Immunol. 10:295-331 (1992); Metcalf, Nature 339:27-30 (1989)). There is also some evidence that members of this receptor superfamily may utilize common tyrosine phosphorylation pathways for signal transduction. Specifically, binding of hematopoietic growth factors to their respective receptors have been found to induce comparable patterns of tyrosine phosphorylation (Ihle, in Interleukins: Molecular Biology and Immunology, Kishimoto, ed., Karger, Basel, pp. 65-106 (1992)).
While it is widely appreciated that cytokine receptors from both families described above play a key role in cellular growth regulation, little is known about the biochemical cascades triggered by the binding of cytokines to these receptors. An understanding of the steps involved in the transduction of the cytokine signal through these receptors would be useful for identifying molecules which play a critical role in signal transduction and which can serve as targets for regulating the activity of these cytokines.
A model for the study of receptor signal transduction has been developed for the erythropoietin receptor (EPOR), one of the hematopoietic growth factor receptors and a member of the cytokine receptor superfamily. Introduction of the EPOR into interleukin-3 (IL-3) dependent cell lines confers on the cells the ability to proliferate in response to EPO (D'Andrea et al., Cell 57:277-285 (1989); Miura et al., Mol. Cell Biol. 11:4895-4902 (1991)). In transfected cells, EPO induces the expression of a series of immediate early genes including c-myc, c-fos, c-pim-1 and egr-1 (Miura et al., Mol. Cell. Biol. 13:1788-1795 (1993)). In addition, EPO induces the rapid tyrosine phosphorylation of a series of cellular substrates (Linnekin et al., Proc. Natl. Acad. Sci. USA 89:6237-6241 (1992); Dusanter-Fourt et al., J. Biol. Chem. 267:10670-10675 (1992); Quelle and Wojchowski, J. Biol. Chem. 266:609-614 (1991); Miura et al., Mol. Cell Biol. 11:4895-4902 (1991); Yoshimura and Lodish, Mol. Cell. Biol. 12:706-715 (1992); Damen et al., Blood 80:1923-1932 (1992)), suggesting that EPOR may function by coupling ligand binding to the activation of a protein tyrosine kinase.
Although the importance of protein tyrosine phosphorylation for biological activities associated with EPO-EPOR binding has been clearly demonstrated, very little has been known concerning the kinases that might be involved. The rapid induction of tyrosine phosphorylation of the carboxyl region of EPOR (Miura et al., Mol. Cell Biol. 11:4895-4902 (1991); Yoshimura and Lodish, Mol. Cell. Biol. 12:706-715 (1992); Dusanter-Fourt et al., J. Biol. Chem. 267:10670-10675 (1992)) suggests that the receptor is closely associated with a kinase, either constitutively or following ligand binding. One study (Yoshimura and Lodish, Mol. Cell. Biol. 12:706-715 (1992)) identified a non-glycosylated protein of 130 kDa that could be cross-linked with the receptor and which was tyrosine phosphorylated either in vivo or in in vitro kinase assays as assessed by its ability to be detected by an anti-phosphotyrosine antibody. Whether the 130 kDa protein was a kinase could not be determined. Recent studies (Linnekin et al., Proc. Natl. Acad. Sci. USA 89:6237-6241 (1992)) also identified a 97 kDa substrate of tyrosine phosphorylation which could be radiolabeled with an azido derivative of ATP, suggesting that it was a kinase. Whether the 130 kDa or 97 Kda potential kinases are previously characterized kinases was not determined.
Tyrosine phosphorylation has also been observed in response to the cytokine interferon gamma (IFNγ). Recent studies (Shuai et al., Science 259:1808-1812 (1992)) have demonstrated that IFNγ induces tyrosine phosphorylation of a 91 kDa protein, and that this 91 kDa protein migrates to the nucleus and binds a γ-activated site.
Tyrosine phosphorylation has further been associated with the response to the cytokine growth hormone (GH). Studies in 3T3-F442A cells showing rapid GH-dependent tyrosyl phosphorylation of multiple proteins, tyrosyl phosphorylation of microtubule-associated protein kinases, and stimulation of microtubule-associated protein kinase activity, as well as the inhibition of these actions by inhibitors of growth hormone receptor (GHR)-associated tyrosine kinase (Campbell et al., J. Biol. Chem. 268:7427-7434 (1993)), suggest a central role for a GHR-associated tyrosine kinase activity in signaling by GH. In addition, the presence of a tyrosine kinase activity in a complex with GH receptor (GHR) prepared from GH-treated fibroblasts has been reported (Carter-Su. et al., J. Biol. Chem. 264:18654-18661 (1989); Stred et al., Endocrinol. 130:1626-1636 (1992); Wang et al., J. Biol. Chem. 267:17390-17396 (1992)). More recently, a nonreceptor tyrosyl phosphorylated 122 kd protein was identified in a kinase-active GH-GHR preparation (Wang et al., J. Biol. Chem. 268:3573-3579 (1993)).
To identify the spectrum of protein tyrosine kinases that are expressed in IL-3-dependent cells which might be involved in signal transduction, polymerase chain reactions (PCR) have been done with degenerative oligonucleotides to conserved protein tyrosine kinase domains (Wilks, Methods Enzymol. 200:533-546 (1991)). Using this approach and Northern blot analysis, IL-3 dependent cells have been shown to express the genes for a number of protein tyrosine kinases including lyn, Tec, c-fes, Jak1 and Jak2 (Mano et al., Oncogene 8:417-424 (1993)). Whether these tyrosine kinases, or other as yet unidentified tyrosine kinases, are involved in cytokine signal transduction is largely unknown.
The potential involvement of lyn kinase in signal transduction was indicated by a recent studies that indicated that IL-3 stimulation increased lyn kinase activity in immune precipitates (Torigoe et al., Blood 80:617-624 (1992)).
Two of the other tyrosine kinases expressed in IL-3-dependent cells, Jak1 and Jak2, belong to the Jak family of kinases. The Jak (Janus kinase; alternatively referred to as just another kinase) family of kinases were initially detected in PCR amplification of tyrosine kinase domains in hematopoietic cells (Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607 (1989)). These studies identified two closely related genes (FD17 and FD22; later termed Jak2 and Jak1) from which the major PCR amplification products were derived. The complete structure of the human Jak1 gene has been reported (Wilks et al., Mol. Cell. Biol. 11:2057-2065 (1991)) and, recently, a partial sequence of the murine Jak2 gene was published (Harpur et al., Oncogene 7:1347-1353 (1992)). Independently a third member of the family (Tyk2) was isolated by screening a cDNA library with a tyrosine kinase domain probe from the c-fms gene (Firmbach-Kraft et al., Oncogene 5:1329-1336 (1990)). The family is characterized by the presence of two kinase domains, one of which is a carboxyl domain that has all the hallmarks of protein kinases. The second domain is immediately amino terminal and bears all the hallmarks of a protein kinase but differs significantly from both the protein tyrosine and serine/threonine kinases. Amino terminal to the kinase domains, there are no SH2 and SH3 domains that characterize most of the non-receptor tyrosine kinases. However, there is extensive similarity in this region among the Jak family members and a number of homology domains have been defined (Harpur et al., Oncogene 7:1347-1353 (1992)).
A link between one member of the Jak family of kinases and the signal transduction of interferon alpha (IFNα) has been recently reported (Velazquez et al., Cell 70:313-322 (1992); Fu, Cell 70:323-335 (1992); Schindler et al., Science 257:809-813 (1992)). Using a genetic approach, the Tyk2 gene was cloned by its ability to functionally reconstitute the cellular response to IFNα in a mutant human cell line that was unresponsive to IFNα. No other link between Tyk2, or any other member of the Jak kinase family, and the signal transduction of any cytokine other than IFNα has been reported.
Ciliary neurotrophic factor (CNTF), as its name implies, is a protein that is specifically required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., J. Neurochem. 34:69-75 (1980)). CNTF has been cloned and synthesized in eukaryotic as well as bacterial expression systems, as described in International Application No. PCT/US90/05241, filed Sep. 14, 1990 by Sendtner et al., incorporated by reference in its entirety herein.
Over the past decade, a number of biological effects have been ascribed to CNTF in addition to its ability to support the survival of ciliary ganglion neurons. CNTF is believed to induce the differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain (Hughes et al., Nature 335:70-73 (1988)). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, Brain Res. 389:39-46 (1986)).
Several novel activities of CNTF have also been discovered, including its ability to support the survival and differentiation of motor neurons and hippocampal neurons, and to increase the rate of hippocampal astrocyte proliferation (International Application No. PCT/US 90/05241, supra).
The CNTF receptor (CNTFR or CNTFRα) has been cloned and expressed in eukaryotic cells, as described in International Application No. PCT/US91/03896, filed Jun. 3, 1991, incorporated herein by reference in its entirety.
The sequence of CNTFR reveals that, unlike most receptors which contain an extracellular domain, a hydrophobic transmembrane domain, and a cytoplasmic domain, CNTFR does not appear to have a cytoplasmic domain. Additionally, the transmembrane hydrophobic domain is proteolytically processed, with the mature form of CNTFR becoming anchored to the cell surface by an unconventional linkage, referred to as a glycophosphatidyl inositol (GPI) linkage (Id.). GPI-linked proteins such as CNTFR may be released from the cell surface through cleavage of the GPI anchor by the enzyme phosphatidylinositol-specific phospholipase C. Of other known receptor sequences, CNTFR is related to a number of receptors, referred to herein as the CNTF/IL-6/LIF receptor family, including IL-6, LIF, G-CSF and oncostatin M (OSM) (Bazan, Neuron 7:197-208 (1991); Rose and Bruce, Proc. Natl. Acad. Sci. 88:8641-8645, (1991)), but appears to be most closely related to the sequence of the receptor for IL-6. However, IL-6 has not been shown to be a GPI-linked protein (e.g., Taga et al., Cell 51:573-581 (1989); Hibi et al., Cell 63:1149-1157 (1989)).
The cloning, sequencing and expression of the CNTF receptor (CNTFR) led to the discovery that CNTFR and CNTF may for a complex that interacts with a membrane bound, signal transducing component, thus suggesting therapeutic activity of a soluble CNTF/CNTFR receptor complex.
One such signal transducing component involved in high affinity binding of CNTF and the subsequent functional response of the cell has been identified as gp130, a β component common to the IL-6, OSM, LIF family of receptors (Fukunaga et al., EMBO J. 10:2855-2865 (1991); Gearing et al., EMBO J. 10:2839-2848 (1991); Gearing et al., Science 255:1434-1437 (1992); Ip et al., Cell 69:1121-1132 (1991)). A further β component identified as being involved in binding and signal transduction in response to LIF (LIFRβ) appears to be the same or similar to a β component necessary for response to CNTF. (As a consequence of the identification of β components necessary for binding and signal transduction of CNTF, what was originally generally termed CNTFR is currently referred to as CNTFRα).
IL-6 is a pleiotropic cytokine which acts on a wide variety of cells, exerting growth promotion and inhibition and specific gene expression sometimes accompanied by cellular differentiation; it has been implicated as being involved in several diseases including inflammation, autoimmunities and lymphoid malignancies (Kishimoto et al., Science 258:593 (1992)). LIF, G-CSF and OSM are all broadly acting factors that, despite having unique growth-regulating activities, share several common actions with IL-6 during hemopoiesis as well as in other processes. For example, all can inhibit the proliferation and induce the differentiation of the murine myeloid leukemia cell line, M1 (Rose and Bruce, Proc. Natl. Acad. Sci. 88:8641-8645 (1991)). LIF and OSM induced tyrosine phosphorylations and gene activation in neuronal cells which are indistinguishable from responses induced by CNTF (Ip et al., Cell 69:1121-1132 (1992)).
Although the events surrounding CNTF binding and receptor activation have recently been elucidated (Davis et al., Science 253:59-63 (1991); Ip et al., Cell 69:1121-1132 (1992); Stahl et al., Cell 74:587-590 (1993); Davis et al., Science 260:1805-1018 (1993)), the mechanism by which signal transduction is initiated inside the cell is more poorly understood. Like the other distantly related receptors for the extended cytokine family—which includes Interleukin (IL)-3, IL-5, GM-CSF, G-CSF, EPO, GH, and the interferons ((Bazan, J. F., Proc. Natl. Acad. Sci. USA 87:6934-6938 (1990); Bazan, J. F., Neuron 7:197-208 (1991))—the CNTF receptor β subunits gp130 and LIFRβ do not have protein tyrosine kinase domains in their cytoplasmic regions (Hibi et al., Cell 63:1149-115 (1990); Gearing et al., EMBO J. 10:2839-2848 (1991)). In spite of this, CNTF-induced dimerization of the β subunits somehow result in the rapid accumulation of a set of tyrosine phosphorylated proteins called the CLIPs (Ip et al., Cell 69:1121-1132 (1992)).
Although, as described above, two of the more prominent CLIPs were identified as the β subunits themselves, most of the others have yet to be characterized. The activation of cytoplasmic tyrosine kinase(s) appears to be essential for CNTF action since inhibitors that block the tyrosine phosphorylations also block subsequent downstream events such as gene inductions (Ip et al., Cell 69:1121-1132 (1992)).
A possible clue to the identity of the cytoplasmic tyrosine kinase(s) activated by the CNTF family of factors came from the finding that other distantly related cytokines resulted in the activation of the Jak/Tyk family of kinases (Firmbach-Kraft et al., Oncogene 5:1329-1336 (1990); Wilks et al., Mol. Cell. Biol. 11:2057-2065 (1991); Harpur et al., Oncogene 7:1347-1353 (1992)). This family of nonreceptor cytoplasmic protein tyrosine kinases consists of 3 known members—Jak1, Jak2, and Tyk2—which are all equally related to each other and share the unusual feature of having two potential kinase domains and no Src homology 2 (SH2) domains. Elegant studies involving complementation of a genetic defect in a cell line unresponsive to IFNa resulted in the identification of Tyk2 as a required component of the IFNα signaling cascade ((Velasquez et al., Cell 70:313-322 (1992)). More recently, the receptors for cytokines such as EPO, GM-CSF, and GH were shown to associate with and activate Jak2 (Argetsinger et al., Cell 74:237-244 (1993); Silvennoinen et al., Proc. Natl. Acad. Sci. USA (1993, in press); Witthuhn et al., Cell 74:227-236 (1993)). The kinase was shown to bind to the membrane proximal cytoplasmic region of the receptor, and mutations of this region that prevented Jak2 binding also resulted in the loss of EPO induced proliferation, suggesting that Jak2 plays a critical role in EPO signaling. Jak1 has not been reported to be significantly activated by any of these receptor systems.
The identification of hemopoietic factors that share receptor components with CNTF would enable the utilization of CNTF and its specific receptor components for activation of targeted cells that are normally responsive to such hemopoietic factors.
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